The Functional Roles of Lactobacillus acidophilus in Different Physiological and Pathological Processes

Probiotics are live microorganisms that can be consumed by humans in amounts sufficient to offer health-promoting effects. Owing to their various biological functions, probiotics are widely used in biological engineering, industry and agriculture, food safety, and the life and health fields. Lactobacillus acidophilus (L. acidophilus), an important human intestinal probiotic, was originally isolated from the human gastrointestinal tract and its functions have been widely studied ever since it was named in 1900. L. acidophilus has been found to play important roles in many aspects of human health. Due to its good resistance against acid and bile salts, it has broad application prospects in functional, edible probiotic preparations. In this review, we explore the basic characteristics and biological functions of L. acidophilus based on the research progress made thus far worldwide. Various problems to be solved regarding the applications of probiotic products and their future development are also discussed.


Fig. 1. Probiotic properties and biological functions of Lactobacillus acidophilus.
L. acidophilus is a species of beneficial microbial flora and has been proven to play an important role in many pathological and physiological processes. It has been shown to improve CVD and lactose intolerance, prevent and treat cancer, regulate immunity, and improve gastrointestinal diseases. with pathogenic bacteria is an important mechanism for L. acidophilus to inhibit the function of pathogenic bacteria, thereby interfering with their invasion into cells [27]. Surface the S-layer protein, extracellular polysaccharide and lipoteichoic acid of many strains can compete for adhesion with pathogenic bacteria [21,28]. Third, the role of L. acidophilus in many diseases also depends on its ability to reduce serum cholesterol level [29,30]. L. acidophilus can absorb and assimilate cholesterol [31,32].
Although some mechanisms have been found, neither these mechanisms nor the influencing factors of L. acidophilus in the host have been fully studied and efforts should be made to explore them further.

Risk Reduction of Cardiovascular Disease
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide, accounting for about one third of global deaths [33]. The occurrence of CVD is related to many factors [34][35][36]. Increasing blood cholesterol is one risk factor that directly impacts CVD. In the 1970s, Mann and Shaper found that populations of particular African tribes generally possessed lower incidences of high serum cholesterol [37]. After investigation, they found that the residents of these tribes regularly drank yogurt fermented by L. acidophilus, suggesting that this bacteria might regulate blood lipids [37]. Since then, more researchers have paid attention to the uses of L. acidophilus and other probiotics relevant to cholesterol reduction and the cholesterol-lowering effects of L. acidophilus have been confirmed. In addition, studies have investigated the effects of L. acidophilus intervention on cholesterol reduction and atherosclerosis development in animal models.
Harrison and Peat reported that the addition of L. acidophilus to baby feed reduced infant serum cholesterol from 147 mg/100 ml on the 5th day down to 119 mg/100 ml by the 8th day of intervention [38]. This decrease in serum cholesterol levels was accompanied by a significant increase in the number of LAB. The number of Escherichia coli present in fecal samples also decreased [38]. Stepankova et al. found that supplementation of L. acidophilus ATCC 4356 also promoted the proliferation of bifidobacteria [39]. Gilliland and Walker showed that L. acidophilus NCFM was able to remove cholesterol from laboratory growth media [40,41]. NCFM has been reported to ingest cholesterol in the presence of bile and in the absence of oxygen, both of which occur in the gut. The researchers tested these effects on young pigs and the results showed that feeding L. acidophilus to pigs significantly inhibited the increased serum cholesterol levels that are usually observed in individuals fed a highcholesterol diet. Park and colleagues have reported that adding L. acidophilus 43121 to a high-cholesterol diet given to rats resulted in reduced serum cholesterol [42,43]. Song et al. showed that L. acidophilus NS1 can reduce plasma LDL-C by increasing the expression of LDLR and SREBP2 in the liver [44].
Huang et al. found that L. acidophilus ATCC 4356 had a significant cholesterol-lowering effect on rats fed with a high-cholesterol diet by inhibiting the expression of NPC1L1 in the small intestine [31,45]. Chen and colleagues found that L. acidophilus ATCC 4356 alleviated atherosclerotic lesions in ApoE −/− mice [46]. L. acidophilus ATCC 4356 inhibited oxidative stress by regulating the production of MDA, oxLDL and SOD, suppressed inflammation via regulations of TNF-α and IL-10 levels, and improved intestinal flora, resulting in blocked progression of atherosclerosis. However, it did not significantly reduce cholesterol levels. In different experimental models, many studies have produced different results due to inconsistent experimental methods. Therefore, the role and functional mechanism of L. acidophilus in reducing cholesterol and alleviating atherosclerosis require further detailed exploration.

Improvement of Gastrointestinal Disease Outcomes
Studies have shown that probiotics can regulate intestinal flora, play a beneficial role in inflammatory diseases such as ulcerative colitis (UC), and have been used effectively to treat and suppress human intestinal infections. Lightfoot and colleagues described the role of L. acidophilus NCFM surface layer protein A as a key effector in the prevention of colitis in mice [47] . Chandhni et al. also showed that the surface proteins in NCFM strains could reverse histopathological damage caused by colitis [48], thus providing a potentially safer option for the treatment of inflammatory bowel disease.
In the normal intestinal flora in humans, L. acidophilus plays a key role in inhibiting the growth of pathogens such as Salmonella enteritidis, Staphylococcus aureus, and Shigella dysenteriae. Therefore, studies have shown that L. acidophilus exhibited strong anti-inflammatory activities [49]. Moshiri et al. reported that L. acidophilus PTCC 1643 could affect the expression of TLR2 and TLR4 in HT29 intestinal epithelial cells under the action of Salmonella enterica serovar Enteritidis (SesE), and inhibit the inflammatory response caused by SesE infection [50]. Small intestinal bacterial overgrowth (SIBO) refers to the changes in the number or types of flora in the small intestine. It is considered to be a condition that can exist for many years without causing obvious symptoms although it is related to chronic digestive problems. Studies by Simenhoff and colleagues have shown that NCFM can inhibit the overgrowth of small intestinal bacteria, reduce the levels of toxic metabolites such as dimethylamine and nitrosodimethylamine in the blood, and positively affect intestinal colonization [51,52], thus improving the nutritional status of patients. These observations support the use of L. acidophilus for the prevention and treatment of intestinal diseases. Further in vivo and in vitro studies are needed to elucidate the detailed mechanisms of these anti-inflammatory effects.

Improvement of Lactose Intolerance
Lactose intolerance, also known as lactose indigestion or lactose malabsorption, refers to the state in which the human body does not produce the enzyme lactase. After consuming milk or dairy products, some people might have diarrhea and other symptoms of intestinal discomfort due to the osmotic effect of the undecomposed lactose.
Previously, many studies have proved that LAB have the ability to be a source of lactase in the small intestine, which helps people with lactase deficiency to digest lactose. Related fermented dairy products may also enhance lactose tolerance [53].
Some probiotic studies have shown that L. acidophilus can improve lactose digestion or symptoms in lactoseintolerant patients [54,55]. Among these studies, in vitro evaluation of the lactase levels of various probiotics has shown that the lactase levels of L. acidophilus NCFM were high when compared to all of the probiotics tested. Multiple studies have also shown that NCFM can improve lactose digestion and relieve symptoms of lactose intolerance such as bloating and diarrhea [56,57]. A study speculated that the bacteria might metabolize lactose during digestion and transport it through the gastrointestinal tract. The study by Pakdaman et al. found that L. acidophilus DDS-1, a unique and edible strain, can improve lactose intolerance symptoms such as diarrhea, cramps, and vomiting [58]. However, a number of studies have shown the opposite effects. For example, Newcomer et al. demonstrated that dairy products containing L. acidophilus NCFM did not significantly improve human lactose intolerance [59][60][61]. The reason for these contradictory results has been associated with the levels of NCFM of L. acidophilus. Therefore, in order to better apply the functional properties of L. acidophilus and to improve lactose intolerance, it is essential to explore and adjust the probiotics levels and the formula with each product.

Prevention and Treatment of Cancer
Probiotics are considered a safe and cost-effective way to prevent or treat a variety of cancers, including colon and liver cancer. Several studies have suggested that consumption of cultured dairy products may reduce colon cancer risk, since the effects of diet are mediated by metabolic effects of intestinal organisms. The activities of βglucuronase, nitroreductase, azoreductase and other microbial enzymes have been used to monitor colon cancer changes. Goldin and Gorbach observed that adding live L. acidophilus into the diet of carnivorous rats significantly reduced azoreductase, nitroreductase and glucuronidase activity [24,25]. The incidence of colon cancer in rats with L. acidophilus NCFM was also lower. Their later study found that NCFM alongside antibiotics inhibited the growth of colon tumors in rats. In human, daily consumption of milk containing NCFM reduced the activity of these three fecal enzymes by a factor of two-to four-fold and reduced the incidence of colon cancer [24,25]. In addition, they found that nitroreductase activity continued to decrease even three weeks after fermented milk intake was stopped, thus indicating a long-term change in colonic flora [24,25].
Studies have shown that the extracellular polysaccharides (EPSs) synthesized by L. acidophilus have exerted health benefits by stimulating the immune response and fighting tumor cells [62]. The anticancer and immunomodulatory activities of EPSs synthesized by L. acidophilus have been proven to combat colon cancer and inflammatory liver cancer. Khedr and colleagues used male rats as a model and confirmed that L. acidophilus ATCC 4356 EPSs had immunomodulatory effects on liver cancer induced by diethylnitrosamine (DEN) and gamma radiation (IR) [63]. They proposed that L. acidophilus ATCC 4356 EPSs might be used as a safe and effective probiotic to prevent and treat liver cancer.

Regulation of Immune Capacity
The immune function of L. acidophilus is mainly conducted via regulating the body's immune system, limiting pathogen colonization within the body, and controlling metabolic disorders and enteritis. Probiotics are used clinically to treat diseases caused by immune system disorders. This can significantly reduce infection time and respiratory tract infection frequency and also improve the therapeutic effects for allergic asthma.
The role of L. acidophilus in regulating the body's ability to respond to immune responses has been demonstrated in previous studies. Wagner et al. confirmed that NCFM induced antibody-and cell-mediated responses to Candida albicans in immunodeficient mice [64]. The serum levels of IgG, IgA, and IgM were higher in euthymic immunocompromised mice and were thought to reduce the severity of candidiasis. Yoghurt prepared with yoghurt cultures containing NCFM, Streptococcus thermophilus, Lactobacillus bulgaricus and Bifidobacterium were tested for their effects on mucosal and systemic IgA and IgG responses in mice immunized orally with cholera toxin. The results showed that IgA against the cholera toxin was higher in the intestine and serum of the mice fed the formulated yogurt than that observed in the mice fed skim milk [65]. These results suggest that coculture including NCFM may increase immune responses to oral antigens.

Other Functions
We summarized the biological functions, processes, and effects of L. acidophilus-related strains in pathological and physiological processes (Table 1). However, the biological functions of L. acidophilus are far more extensive than what we have mentioned. The role of L. acidophilus in many other diseases is constantly being explored and new discoveries are being made. Studies have shown that kidney tissue damage can be alleviated by reducing oxidative stress, inflammation, and cell death [66]. Zhang et al. first explored the relationship between ATCC 4356 and renal ischemia-reperfusion injury (IRI) [67]. They found that L. acidophilus ATCC 4356 alleviated renal IRI through antioxidant stress and anti-inflammatory responses and improved intestinal microbial distribution in renal IRI mice. In the following treatment with ATCC 4356, the levels of anti-inflammatory factors (IL-4 and IL10) were upregulated, whereas the levels of pro-inflammatory factors (IL-1β, IL-8, TNF-α and IFN-γ) were downregulated. In addition, renal tissue apoptosis in IRI mice was reduced [67].
Studies have shown that oral L. acidophilus can improve heart function in mice with myocardial infarction. Sadeghzadeh and co-researchers found that L. acidophilus was able to improve the hemodynamic and histopathological indicators of the ISO-induced myocardial injury rat model [68], providing obvious myocardial protection. In the future, probiotic supplements may become a new option for patients with ischemic heart disease.
L. acidophilus has been shown to have potential applications in the prevention and control of genitourinary and vaginal infections. Reid et al. precultured L. acidophilus NCFM with urinary and vaginal epithelial cells from healthy women and subsequently exposed them to different urinary tract pathogens. Results showed that NCFM competitively excluded these pathogens and effectively prevented and suppressed urinary tract and vaginal infections [69].
Rheumatoid arthritis (RA) is a common inflammatory joint disease. It has been reported that the ingestion of L. acidophilus ATCC 314 exerted anti-inflammatory and potent antioxidant properties in a collagen-induced arthritis (CIA) rat model [70,71]. This suggests that L. acidophilus is a promising treatment that should be tested further in RA patient preclinical trials.

Applications and Future Prospects of L. acidophilus
As people pay more and more attention to health issues, it is of great importance that different kinds of probiotics within food are able to play a healthy role. Of these probiotics, L. acidophilus is one of the most commonly used microorganisms, as it is thought to have various beneficial effects on human health. These advantageous effects include lowering blood cholesterol, improving gastrointestinal diseases, and reducing the risk of lactose intolerance and carcinogenicity [72,73]. Research and development into L. acidophilus has received widespread attention. It is referred to as the third-generation yogurt starter strain. L. acidophilus has good acid and bile salt resistance and produces a variety of antibacterial substances during metabolism. Due to these attributes, L. acidophilus strains have broad application prospects as functional, edible bacteria.
Despite these favorable characteristics, through our analysis of studies on L. acidophilus which highlighted our present understanding of the current application status, we found that there are also many problems and limitations in the research and application of L. acidophilus. First, due to different naming methods, the same Inhibit CVD progression Inhibit oxidative stress by modulating the productions of MDA, oxLDL and SOD; suppress inflammatory status by regulating TNF-α and IL-10 levels; inhibit NPC1L1 expression in the small intestine; improve intestinal microflora; inhibit the development of atherosclerosis. [31,40,45,46] L. acidophilus NCFM Assimilate cholesterol and control cholesterol levels. [40,41] L. acidophilus 43121 Affect cholesterol metabolism and reduce blood cholesterol levels. [40,42,43] L. acidophilus NS1 Reduce plasma LDL-C by increasing hepatic LDLR and SREBP2 expression. [44] L. acidophilus NCFM

Improve gastrointestinal diseases
Strain surface layer proteins play an important role; alleviate Tcell-induced colitis by significantly reducing the proinflammatory response; preserve microbiome composition and intestinal barrier function; reverse histopathological damage caused by colitis; reduce the level of toxic metabolites. [47,48,51,52] L. acidophilus PTCC 1643 Modulate the expression of TLR2 and TLR4 in HT29 intestinal epithelial cells challenged with SesE; enhance anti-inflammatory effects. [50] L. acidophilus NCFM Improve lactose intolerance Strain has a higher level of lactase, which metabolizes lactose during digestion and transits through the gastrointestinal tract, thereby improving lactose digestion. [56,57] L. acidophilus DDS-1 Assist in breaking down lactose; improve lactose intolerance symptoms such as diarrhea, cramps and vomiting. [58] L. acidophilus ATCC 4356 Prevent and treat colon cancer, liver cancer and other cancers The exopolysaccharides of the strain have immunomodulatory and antitumor activities; regulate the TLR2/STAT-3/P38-MAPK pathway associated with inflammation against HCC. [63] L. acidophilus NCFM Stimulate the immune response; reduce the activities of βglucuronase, nitroreductase, azoreductase and other microbial enzymes; produce compounds that inhibit tumor proliferation; reduce the incidence of colon cancer and inhibit the growth of colon tumors. [24,25] L. acidophilus NCFM

Regulate immune capacity
Reduce levels of pro-inflammatory cytokines significantly and mobilize a systemic immune response; limit pathogen colonization in the body, control metabolic disorders. [47,64,65] L. acidophilus ATCC314

Manage inflammatory disorders
Regulate the secretion of inflammatory cytokines; reduce oxidative stress. [70] strain may have multiple names. This may lead to confusion during literature searches and research. Important findings could be missed, resulting in incomplete information collection. Second, due to the different research methods used to investigate probiotics, results from different research groups may deviate from each other. Different concentrations and ratios of probiotics have been shown to have different effects, therefore the health benefits of certain disease symptoms remain to be proven. Further research on the optimal strains, doses and dosing algorithms are of key importance for future research. In addition, different species of L. acidophilus can exhibit similar probiotic effects in vitro, but their properties differ significantly when evaluated in vivo. Currently, probiotic regulation of intestinal flora is recognized as an interesting way to prevent certain diseases. Recent studies have proposed many mechanisms by which probiotics function, but the effectiveness of many probiotics has not been proven in different conditions, which has presently limited the promotion and application of probiotics. There are probably several main reasons why these research limitations occur, including too many low-quality studies, variability within the microbiome, and great diversity between the probiotic strains used. However, some studies have reported reasonable and encouraging results that support further research into probiotics. With this in mind, we should put more effort into overcoming the difficulties. First, because most studies have, so far, focused on animal studies or small human research groups, it is difficult to assess the possible health effects of these probiotics in the general population. Therefore, we need to conduct more extensive epidemiological evaluations which take into account the variability between patients. Due to the high cost of such interventions, it is necessary to characterize strains well to select the strains that are most effective for a particular application. Second, we should identify bacterial markers of the microbiome in related diseases in order to gain sufficient clinical trial capacity. Furthermore, new methods for analyzing the microbiome and its function will greatly facilitate the research when studying large numbers of samples. Probiotics could serve as a low-cost, lowrisk alternative to antibiotic treatment in order to prevent infection. We believe that the development of probiotics will open up another impressive field of research. Further research in this area may provide exciting avenues for healthcare strategies, as well as creating more economic and social benefits.